Progressive hearing loss is a common human health problem that often stems from the loss or dysfunction of sensory hair cells in the inner ear. Actin proteins are central to hair cell structure and function; consequently, understanding the molecular mechanisms of cytoskeletal maintenance is a key step in considering rationally designed therapies for age-related or noise-induced hearing loss. -Actin and ?-actin, two distinct yet closely related actin isoforms, each contribute to the population of filamentous actin in stereocilia. We have previously shown that mice with hair cells lacking either actin isoform form normal stereocilia, but go on to develop different forms of progressive hearing loss. This suggests that -Actin and ?-actin each make unique contributions to stereocilia stability. This proposal is focused on two aspects of actin biology in stereocilia. In Aim 1, we will use existing conditional -Actin and ?-actin knockout mouse lines and a newly developed transgenic actin reporter line to assess actin dynamics in stereocilia in vivo. In Aim 2, we will use cell biologica and biochemical approaches to determine if differential interactions between the actin bundling protein fascin 2 and -actin and ?-actin contribute to distinct stereocilia maintenance mechanisms. Together, completion of these Aims will support a new model of actin regulation during stereocilia maintenance. In the long term, we will use the same suite of universal platforms established in this proposal to assess the roles of other deafness-causing mutant actin binding proteins in modulating stereocilia actin dynamics at the level of molecules, cells and mice. PUBLIC HEALTH RELEVANCE: Age-related hearing loss is the most common form of hearing impairment in humans and is often caused by degeneration of specialized sensory cells in the inner ear. Many of these cells depend on different kinds of actin proteins for normal structure and function. We are proposing to elucidate the mechanisms through which each type of actin contributes to cell maintenance to provide the foundation for rationale design of therapeutic interventions.